Project Summary/Abstract Gene expression is regulated at multiple layers, from histone modifications (histone code) through RNA processing to protein degradation. Most layers are extensively studied because of their fundamental importance to biology and because dysregulation at each layer can lead to diseases. However, the regulatory role of specialized ribosomes (ribosome code) is largely unexplored because, despite decades of research, the evidence that ribosomes can actively regulate gene expression had remained indirect and inconclusive; multiple studies have shown that mutations in core ribosomal proteins (RPs) have highly RP-specific phenotypes in processes including cell differentiation, aging and carcinogenesis. These correlative associations and my observations of differentiation RP transcription motivated me to directly test whether wildtype cells build specialized ribosomes. To this end, I developed methods for direct, accurate and precise measurements of protein levels and synthesis rates. These methods allowed me to obtain the first direct evidence for differential stoichiometry among RPs in unperturbed yeast and mammalian stem cells. Here I propose to substantially expand this methodology and use it to investigate ribosome-mediated translational regulation in the context of differentiating mouse embryonic stem cells (ESC). Previous research has indicated that knockdowns of some RPs can specifically prevent stem-cell differentiation but not self-renewal and these observations dovetail with my preliminary data indicating change in the ribosomal composition during ESC differentiation. I propose to build upon these results by: (i) employing and expanding my newly-developed methodology for building the first comprehensive map of ribosome modifications, (ii) mapping specific interactions between mRNAs and ribosome modification, (iii) associating these interactions with functional impact on the rates of translation initiation and elongation, and on cell differentiation, and (iv) testing causal mechanisms suggested by these associations by genetic manipulations of RP. The proposed methods and experiments have the potential to overcome longstanding roadblocks and to lay the groundwork for understanding ribosome-mediated translational regulation.